Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61N—ELECTROTHERAPY; MAGNETOTHERAPY; RADIATION THERAPY; ULTRASOUND THERAPY
  • A61N5/00—Radiation therapy
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/02—Bacterial antigens
  • A61K39/04—Mycobacterium, e.g. Mycobacterium tuberculosis
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61N—ELECTROTHERAPY; MAGNETOTHERAPY; RADIATION THERAPY; ULTRASOUND THERAPY
  • A61N1/00—Electrotherapy; Circuits therefor
  • A61N1/18—Applying electric currents by contact electrodes
  • A61N1/20—Applying electric currents by contact electrodes continuous direct currents
  • A61N1/205—Applying electric currents by contact electrodes continuous direct currents for promoting a biological process
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61N—ELECTROTHERAPY; MAGNETOTHERAPY; RADIATION THERAPY; ULTRASOUND THERAPY
  • A61N1/00—Electrotherapy; Circuits therefor
  • A61N1/18—Applying electric currents by contact electrodes
  • A61N1/32—Applying electric currents by contact electrodes alternating or intermittent currents
  • A61N1/325—Applying electric currents by contact electrodes alternating or intermittent currents for iontophoresis, i.e. transfer of media in ionic state by an electromotoric force into the body
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61N—ELECTROTHERAPY; MAGNETOTHERAPY; RADIATION THERAPY; ULTRASOUND THERAPY
  • A61N1/00—Electrotherapy; Circuits therefor
  • A61N1/18—Applying electric currents by contact electrodes
  • A61N1/32—Applying electric currents by contact electrodes alternating or intermittent currents
  • A61N1/36—Applying electric currents by contact electrodes alternating or intermittent currents for stimulation
  • A61N1/36014—External stimulators, e.g. with patch electrodes
  • A61N1/36017—External stimulators, e.g. with patch electrodes with leads or electrodes penetrating the skin

Definitions

• the present invention is related to a method for making skeletal muscle semipermeable to pharmaceutical drugs and nucleic acids More specifically, skeletal muscle is made semipermeable by electrically stimulating the muscle at low field strengths following pharmaceutical drugs and nucleic acids injection
• Skeletal muscle is a promising candidate for drug delivery, gene therapy and genetic immunization
• skeletal muscle constitutes over 50% of a human's body mass, most of which is easily accessible compared to other tissues and organs of the body
• DMD Duchenne muscular dystrophy
• diabetes mellitus a malignant neoplasm originating from the muscle
• hyperlipidaemia a malignant neoplasm originating from the muscle
• cardiovascular disease which are good candidate disorders for drug and gene delivery into the muscle
• muscle is an ideal site for genetic immunization because it is easily accessible and proteins made in the muscle are secreted, thus eliciting an immune response.
• skeletal muscle cells are non-dividing Therefore,
• C0NFIRMATI0N COPY skeletal muscle cells are capable of expressing a protein coded by a gene for a longer time period than would be expected of other cell types that are continually dividing Because the protein is expressed for a longer time, fewer treatments would be necessary
• the present invention provides a method for delivering or transfecting pharmaceutical drugs and DNA into skeletal muscle Without being bound by theory, the method is thought to be similar to electroporation Electroporation works on the principle that cells act as an electrical capacitor generally unable to pass current Subjecting cells to a high-voltage electric field, therefore, creates transient permeable structures or micropores in the cell membrane These pores are large enough to allow pharmaceutical drugs, DNA and other polar compounds to gain access to the interior of the cell With time, the pores in the cell membrane close and the cell once again becomes impermeable Conventional electroporation, however, employs high field strengths from 0 4 to several kV/cm.
• the field strength used in the present invention ranges from about 25 V/cm to 250 V/cm. These lower field strengths are thought to cause less muscle damage without sacrificing, and indeed increasing, transfection efficiencies. Furthermore, using the method of the present invention, transfection efficiencies can be tightly regulated by altering such parameters as frequency, pulse duration and pulse number.
• the transfection method of the present invention can be used, for example, to transfect expression vectors for genetic immunization (i.e., DNA vaccines).
• rabbits were transfected with a plasmid containing the cDNA for rat agrin
• the transfected muscles produced and secreted agrin protein
• rabbit serum contained significant antibodies against rat agrin
• mice and rats were transfected using the method of the present invention with one or more of three different eukaryotic expression vectors containing the coding sequences for DH-CNTF, an agonistic variant of human ciliary neurotrophic factor, AADH-CNTF, an antagonistic variant of human ciliary neurotrophic factor and sec-DHCNTF, a secreted form of DH-CNTF.
• the muscles were either not electrically stimulated or stimulated immediately after DNA injection Blood was collected at various time points and the antibody titers determined. In both rats and mice, electrical stimulation immediately after DNA injection led to approximately 5 to 10-fold higher antibody titers than simple DNA injection.
• the transfection method of the present invention can also be used to systemically deliver proteins to treat diseases.
• a DNA plasmid harboring the erythropoietin (EPO) gene was injected into skeletal muscle and stimulated according to the method of the present invention Controls were either not stimulated or transfected with a control vector not harboring the EPO gene After 14 days, only the mice transfected with EPO according to the method of the present invention displayed an increased hematocrit indicating the transfected muscles were able to produce and secrete into the blood stream substantial amounts of EPO. Non-nucleic acids may also be transfected by the method of the present invention.
• rhodamin conjugated dextran was injected into the muscle followed by electrical stimulation. Three to five days later the muscles were frozen in liquid nitrogen and sectioned on a cryostat. Fluorescence was observed in cells injected and stimulated, indicating the rhodamin conjugated dextran was able to enter and remain in the muscle cells.
• Figure 2 - is a graphical illustration of an electrical stimulation delivered according to the method of the present invention.
• Figure 3 - illustrates whole mounts of muscles which have been injected with 50 ⁇ l of RSV-Lac Z Plasmid DNA solution at a concentration of 1 ⁇ g/ ⁇ l. Muscles in 3a and 3b were taken out 15 days after DNA injection. Muscles in 3c and 3d were taken out 7 days after DNA injection. All muscles are pairs from the same rat.
• Figure 4 pictures a whole muscle and a 1 mm slice of a transfected muscle. Dark stain indicates o-nitrophenyl-b-D-galactopyranoside (ONPG) that has been catalyzed by ⁇ - galactosidase in the muscle to yield a dark precipitate. Arrows illustrate muscle fibers that were successfully transfected using the method of the present invention.
• ONPG o-nitrophenyl-b-D-galactopyranoside
• Figure 5 - includes mean number of transfected fibers from each group of skeletal muscles shown in Figure 3.
• Figure 6 is a bar graph illustrating mean transfected fibers of muscles from several different experiments and several different batches of DNA grouped together.
• SOL S and EDL S the muscles (16 in each group) have been stimulated directly after the injection of DNA.
• SOL NS and EDL NS the muscles (10 in each group) have been stimulated by the nerve, not stimulated at all or stimulated directly 10 minutes before the DNA injection
• Figure 7 - is a graph illustrating the number of skeletal muscle fibers transfected versus the log of the stimulation frequency The duration of the stimulation train was kept constant at 1 second
• Figure 8 - is a photograph of transfected muscles from which data in Figure 7 were generated.
• Figure 9 - illustrates the results achieved when whole mounts of muscles were transfected according to the method of the present invention using two different electrodes
• Figure 10 - is a graph illustrating the number of skeletal muscle fibers transfected with increasing frequency compared to increasing pulse number
• Figure 11 - is a graph illustration of the number of skeletal muscle fibers transfected versus the number of pulses at constant frequency
• Figure 12 - is a graph illustrating mean luciferace activity versus the number of pulses
• Figure 13 - is a graph illustrating the voltage dependency of the stimulation method of the present invention
• Figure 13a illustrates the luciferase activity of muscle stimulated with varying volts
• Figure 13b illustrates the mean luciferace activity of muscles stimulated with an amplitude above 13 volts and below 5 volts
• Figure 14 - is a graph illustrating the effect of pulse duration on the transfection efficiency
• Figure 15 - is a bar graph illustrating a comparison of transfection efficiencies for varying pulse durations and pulse numbers
• Figure 16 - is a bar graph illustrating the effect of DNA concentration on transfection efficiency
• Figure 17 - is a photograph of transfected muscles illustrating damage caused by stimulation and regeneration of the muscle after a short period of time
• Figure 17a illustrates an injected muscle that was not stimulated
• Figure 17b illustrates muscle damage following muscle stimulation
• Figure 17c illustrates muscle stimulated under harsher stimulation conditions
• Figure 17d illustrates that muscles stimulated under the conditions of muscles in 17c are completely regenerated and repaired after 14 days
• Figure 17e illustrates muscles transfected with green fluorescent protein (GFP)
• Figure 17f illustrates that transfected fibers can bee seen in the vicinity of the damaged area
• Figure 18 - is a photograph of cells stained with anti-agrin polyclonal antibodies derived from a rabbit genetically immunized with an expression vector coding for rat agrin using the stimulation technique of the present invention.
• GFP green fluorescent protein
• Figure 19 - are graphs illustrating improved genetic immunization of mice and rats using the stimulation technique of the present invention versus naked DNA injection.
• Figure 20 - is a photograph of muscles transfected with rhodamine-conjugated dextran and green fluorescent protein. Top: rhodamin fluorescence from rhodamine conjugated dextran. Middle: The same section as above but with filters revealing GFP fluorescence. Bottom: hematoxilin and eosin staining of a neighboring section.
• the present invention is directed to a novel method for increasing the permeability of skeletal muscle tissue, thus allowing pharmaceutical drugs and nucleic acids to enter or transfect the cells.
• the method of the present invention passes a predetermined amount of electrical current through the skeletal muscle tissue.
• the parameters of the method of the present invention are unique, particularly with respect to the low field strength used and the amount of damage that occurs.
• Other parameters such as the number of trains, frequency, pulse number and pulse duration can be varied in order to regulate the amount of pharmaceutical drug or nucleic acid delivered.
• skeletal muscle is exposed and a predetermined amount of a molecule is injected into the muscle.
• the DNA is dissolved in 0.9% sodium chloride (NaCl).
• NaCl sodium chloride
• the exact solvent is not critical to the invention.
• other solvents such as sucrose are capable of increasing DNA uptake in skeletal muscle.
• Other substances may also be co-transfected with the molecule of interest for a variety of beneficial reasons.
• PI 88 Lee, et al. PNAS., 4524-8, 10, 89 (1992)
• seal electropermeabilized membranes may beneficially affect transfection efficiencies by increasing the survival rate of transfected fibers.
• electrodes are placed on the muscle, about 1 -4 mm apart, near the area where the molecule was injected.
• the exact position or design of the electrodes is not critical so long as current is permitted to pass through the muscle fibers perpendicular to their direction in the area of the injected molecule.
• the muscle is electroporated or stimulated As illustrated in Figure 2, the stimulation is delivered as a square bipolar pulse having a predetermined amplitude and duration
• these parameters have been widely varied and transfection efficiencies compared
• the voltages have ranged from approximately 0 to 50 volts; the pulse durations have ranged from 5 ⁇ s to 5 ms; the number of pulses have ranged from a single pulse to 30,000 pulses, and the pulse frequency within trains have ranged from 0 5 Hz to 1000 Hz
• rat soleus or EDL muscles were injected with DNA plasmid containing the ⁇ -galactosidase gene (lac Z)
• the ⁇ -galactosidase gene yields a protein capable of converting a colorless substrate into a blue substrate that can be visually analyzed or measured spectrophotometrically
• Figure 3 depicts representative soleus and EDL muscles that have been transfected with ⁇ - galactosidase gene using various stimulation parameters
• FIG. 3a illustrates the improved DNA delivery efficiency of soleus and EDL muscles that have been transfected according to the method of the present invention.
• the muscles were injected with the ⁇ - galactosidase gene as described above. After the DNA injection, the muscles were either untreated or, immediately after the DNA injection, the muscles were stimulated according to the method of the present invention.
• the method of the present invention was performed on innervated (sciatic nerve not transected) and denervated (sciatic nerve transected) soleus and EDL muscles as described above.
• innervated and denervated muscles produced a generous quantity of blue product indicating high efficiency transfer of the ⁇ -galactosidase gene.
• quantitation of transfected muscle fibers confirms high efficiency transfection of both innervated and denervated muscles.
• transfection efficiency in muscles stimulated via the nerve should yield similar efficiencies as direct muscle stimulation.
• direct nerve stimulation did not significantly increase transfection efficiencies compared to direct muscle stimulation.
• Figure 5c in both soleus and EDL muscles a 10-fold increase in transfection efficiency was observed with direct muscle stimulation.
• the electrical stimulator used for the experiments was manufactured by FHC (Brunswick, ME 04011) Both Pulsar 6bp and the Pulsar 6bp-a/s stimulators have been used
• the Pulsar 6bp-a/s delivers a maximal voltage is 150 V and a maximal current of 50 mA
• the maximal voltage that can be delivered requires a resistance between the electrodes of greater than 3000 ohms
• the stimulators have been operated at constant voltage mode Because of the low resistance in the muscle, the voltages have been lower as discussed in the Examples below In all experiments the current has been maintained at 50mA
• Figure 9 illustrates the results obtained using two different electrodes configuration
• the electrode shown in (A) was placed perpendicular to the muscle fibers It consisted of a silver wire with diameter (d) of 0 6 mm, (C) (this is the electrode which was used in all experiments except in (B))
• One electrode was placed on each side of the muscle A short segment in the middle third of the muscle is positive for the Lac Z staining (A), indicating localized expression
• D The electrode was penetrated into the muscle in parallel with the muscle fibers
• One of the two wires of the electrode was penetrated into the muscle parallel with the muscle fibers
• the second wire was placed on the muscle surface, also parallel with the fibers
• Both types of electrodes ( Figures 9c and 9d) gave a
• the pSV40-luc construct was used in four (4) muscles. It was injected into the soleus muscle, 3 days after the muscles were removed and luciferase activity was measured using the Promega Luciferase Assay System (Daviset et al., 1993) Uninjected EDL from the same rats were used as control.
• nucleic acid can be used with the method of the present invention, for example, plasmid DNA, linear DNA, antisense DNA and RNA
• the nucleic acid is a DNA expression vector of the type well known in the art
• an expression vector contains a promoter operably linked to a DNA molecule that codes for the protein of interest followed by a termination signal such as a polyadenylation signal.
• a termination signal such as a polyadenylation signal.
• Other elements required for bacterial growth and proper mammalian processing may be included, such as the ⁇ -lactamase coding region, an fl origin and ColEl -derived plasmid replication origin. Similar constructs containing a DNA coding region of interest can be constructed by one skilled in the art
• nucleic acid and proteins can be simultaneously introduced into an electroporated muscle In one embodiment, the large
• T-antigen nuclear localization signal was mixed with a plasmid containing the DNA coding region for Lac Z.
• the large T-antigen nuclear localization signal is a protein that binds DNA and facilitates its transport into the nucleus of a cell
• large T-antigen nuclear localization signal has been shown to increase transfection efficiency Using the method of the present invention, large T-antigen nuclear localization signal also increased the transfection efficiency of Lac Z indicating that the protein was able to bind the DNA and enter the muscle cell. 6 EXAMPLES
• Soleus muscles of Wistar rats (200-270 grams) were injected with 50 ⁇ g of RSV luciferase DNA plasmid in 50 ⁇ l 0 9% NaCl.
• the muscles were electrically stimulated using the following parameters 1000 Hz, between 0 - 1000 bipolar pulses of 200 ⁇ s duration in each train were applied to the muscle 30 times over a period of 1 minute Muscles were removed 3 days after transfection and frozen in liquid nitrogen Cryostat sections were taken from the of the muscles and stained with Hematoxolin, Eosin and Safran (see Example 9) The remaining pieces were homogenized as described in Example 4 below As illustrated in Figure 10-12, transfection efficiency increased with the number of pulses delivered to the muscle
• EDL and soleus muscles of Wistar rats were injected with 25 ⁇ g of RSV driven luciferace plasmid DNA in 50 ⁇ l 0 9% NaCl
• the injected muscles were electrically stimulated using the following parameters 100 Hz, 100 bipolar pulses in each train of 200 ⁇ s duration, voltage varied from between 0 to 47 5 Muscles were removed 4 days post injection and stimulation, homogenized in Promega (Madison, WI) luciferace assay buffer and luminescence was measured according to manufacturer's protocols Macintosh computer and a LabWiev acquisition program were used to capture the first voltage pulses Recordings were done in parallel with the stimulation electrodes The voltage measurements were done manually on prints as the average of the maximal voltage of 10 pulses approximately 100 ms after onset of stimulation
• Soleus muscles of Wistar rats (200-270 grams) were injected with 50 ⁇ g of DNA plasmid containing the ⁇ -galactosidase gene in 50 ⁇ l 0 9% NaCl
• the muscles were electrically stimulated using the following parameters- 100 Hz, 25 volts, 100 bipolar pulses in each train having pulse durations ranging from 5-200 ⁇ s
• the number of transfected fibers were counted in a 1 mm thick section from the middle of the muscle under a dissection microscope
• a second set of rats were injected with 25 ⁇ g of RSV-driven luciferace plasmid DNA in 50 ⁇ l 0.9% NaCl and electrically stimulated with the same parameters as above except that the pulse durations were varied from 50-2000 ⁇ s
• the optimal pulse duration ranged from about 50 ⁇ s to about 200 ⁇ s. This method can be used to optimize the pulse duration of other stimulation parameters
• Soleus muscles of six Wistar rats were injected with 50 ⁇ g of DNA plasmid containing the ⁇ -galactosidase gene in 50 ⁇ l 0 9% NaCl
• the muscles were electrically stimulated as described above except that the pulse duration was varied.
• the following electroporation parameters were compared (1) 100 pulses of 50 ⁇ s duration versus 1 pulse of 5000 ⁇ s, and (2) 10 trains of 100 pulses of 50 ⁇ s versus 10 pulses of 5000 ⁇ s.
• Muscles were removed 14 days later and sectioned on a cryostat Cross sections were stained as previously described The number of transfected fibers were counted As illustrated in Figure 15, longer pulse durations result in higher transfection efficiency
• EDL muscles of six Wistar rats were injected with either l ⁇ g ⁇ l or 5 ⁇ g/ ⁇ l of DNA plasmid containing the ⁇ -galactosidase gene in 50 ⁇ l 0.9% NaCl.
• the muscles were electrically stimulated with 30 trains of 100 pulses of 200 ⁇ s duration or not stimulated at all. Muscles were removed 14 days later and sectioned on a cryostat. Cross sections were stained as previously described and transfected fibers were counted. As illustrated in Figure 16, greater transfection efficiencies were obtained with higher DNA concentrates.
• Wistar rat muscles were injected with DNA plasmid containing the ⁇ -galactosidase gene containing a 100: 1 molar excess of large T-antigen nuclear localization signal. This has been shown in other transfection studies to improve the transfection. (See, P. Collas et al. Transgenic Res., 6: 451-8 (1996)).
• the muscle were stimulated with 10 trains of 100 pulses of 50 ⁇ s duration.
• the muscles containing the large T-antigen nuclear localization signal had the highest number of transfected fibers. Specifically, the muscle co-transfected with large T-antigen nuclear localization signal had 100 and 38 transfected fibers versus 7.3 and 4.7 for the muscles transfected only with DNA, respectively.
• Example 9 Muscle Damage Resulting from Stimulation: Muscles from Example 3 that were sectioned and stained to assess the muscle damage from electroporation. As illustrated in Figure 17a, some damage can occur with injection alone, although the majority of unstimulated muscles were undamaged. In muscles stimulated with 300 pulses, more damage was observed ( Figure 17b). As illustrated in Figure 17c, muscle stimulated with 30 trains of 1000 pulses displayed greater damage, indicating that damage is proportional to the extent of stimulation. Figure 17d illustrates that muscles stimulated under the conditions of muscles in 17c are completely regenerated and repaired after 14 days.
• FIG. 17e illustrates muscles transfected with GFP Transfected fibers can bee seen in the vicinity of the damaged area ( Figure 17f). Transfected regenerating fibers were never observed in cross sections 3 days after electroporation
• a female rabbit (4 5 kg) was injected into the right femuralis rectus with 2 milliliters of l ⁇ g / ⁇ l of DNA plasimd containing the rat neural agrin cDNA driven by the CMV promotor (Cohen et al. MCN, 9, 237-53 (1997))
• the first milliliter was injected equally in ten places superficial in the muscle followed by 10 trains of 1000 pulses delivered at a frequency of 1000 Hz
• the second milliliter was placed further down in the muscle
• Muscles were taken out 5 days after transfection and the COS cells were stained 4 days after transfection
• FIG. 18a illustrates the agrin transfected COS cells stained with antiserum from immunized rabbit (diluted 1 100) and fluorescein conjugated secondary antibody COS cells were stained first fixing the cells in 1.5% paraformaldehyde for 10 minutes, followed by a 30 minute wash with phosphate buffered saline (PBS) The cells were then blocked with 0 2% bovine serum albumin, triton X-100, 0 1% in PBS 0 1M, for 4 minutes Serum diluted in same solution was added to the cells and allowed to incubate for 20 minutes Cells were wash for 4 minutes in PBS and incubated with the secondary antibody (Cappel, 55646) for 10 minutes followed by a PBS wash Mouse primary mAb Agr-86 was included in the same antibody mixture and rhodamin conjugated secondary antibody (Sigma T-5393,
• Example 11 Genetic Immunization of Mice: Groups of two-month old male Sprague Dawley rats were inoculated bilaterally in the EDL and soleus muscles with a total of 200 micrograms (4 x 50 microliters of a 1 mg/ml solution of DNA in saline) of three different eukaryotic expression vectors containing the cytomegalovirus immediate early promoter (CMV) and the coding sequences for the following proteins: DH-CNTF, an agonistic variant of human ciliary neurotrophic factor (Saggio et al. EMBO J. 14, 3045-3054, 1995); AADH-CNTF, an antagonistic variant of human ciliary neurotrophic factor (Di Marco et al. Proc. Natl.
• CMV cytomegalovirus immediate early promoter
• sec-DHCNTF a secreted form of DH-CNTF.
• the muscles were either not electrically stimulated or stimulated immediately after DNA injection using 30 trains of 100 or 1000 square bipolar pulses (duration 200 microseconds; amplitude setting 150 V, effective voltage ⁇ 25 V) each, delivered at a frequency of 1000 Hz with a two second interval between successive trains.
• mice Groups of two-month old male CD1 mice were inoculated bilaterally in the quadriceps muscles with 100 micrograms (2 x 50 microliters of a 1 mg/ml solution of DNA in saline) of sec-DHCNTF plasmid, with or without electrical stimulation of the muscle immediately after DNA injection.
• Stimulation conditions were 10 trains of 1000 square bipolar pulses (amplitude setting 150 V) delivered at a frequency of 1000 Hz with a two second interval between successive trains.
• Example 12 Secreted Proteins with Systemic Biological Activity: Fifty micrograms (50 microliter of a 1 mg/ml solution in 0 9% NaCl) of a eukaryotic expression plasmid (CMV-EPO) containing the cDN A of mouse erythropoietin under the control of the cytomegalovirus immediate early promoter was injected in the left quadriceps muscle of three-month old 129xBalb/C female mice In five mice (group 1 ), the muscles were electrically stimulated immediately after DNA injection using 10 trains of 1000 square bipolar pulses of 200 microseconds duration, with an interval of 2 seconds between successive trains The frequency of the trains was 1000 Hz and the amplitude set at 150 V (effective voltage -25 V).
• CMV-EPO eukaryotic expression plasmid
• mice In another group of 5 mice (group 2) the muscles were not stimulated after DNA injection
• group 3 a group of 4 mice (group 3) was injected with a plasmid (CMV-GFP) containing the coding sequence for green fluorescence protein under the control of the CMV promoter, followed by electrical stimulation at the same conditions as group 1
• Group 4 consisted of 5 mice injected only with saline solution without electrical stimulation
• mice injected with the EPO construct and electrically stimulated immediately thereafter Serum samples were analyzed for the presence of EPO using a commercial ELISA kit (R&D Systems) The results are shown in Table 4 In all groups of mice, except those that were injected with the EPO construct and electrically stimulated immediately thereafter, circulating EPO levels were below the limit of detection of the ELISA kit ( ⁇ 15 mU/ml) In contrast, mice injected with the EPO construct and electrically stimulated had significantly elevated serum EPO levels 5 days after injection (average of approximately 50 mU/ml) The serum concentration of EPO remained elevated for up to 28 days following DNA injection (latest time point examined, data not shown) These levels of EPO produced an increase in hematocrits, which rose from 46 2% prior to injection to 70 0% and 76 7% at 14 and 28 days after DNA injection, respectively These values were significantly different from those obtained with both control groups (groups 3 and 4) and from those of mice injected with
• ND not determined a p ⁇ 0 0001 vs group 2, b p ⁇ 0 0001 vs group 3, c p ⁇ 0 0001 vs group 4 (Fisher's protected least significant difference)
• the injection procedure and stimulation pattern (100 Hz) was the same as previously described.
• the electrode shown in (A) was placed perpendicular to the muscle fibers. It consisted of a silver wire with diameter (d) of 0.6 mm, (C) (This is the electrode which was used in all experiments except in (B) ) .
• One electrode was placed on each side of the muscle. A short segment in the middle third of the muscle is positive for LacZ staining (A) , indicating localised expression.
• the electrode was penetrated into the muscle in parallell with the muscle fibers. A second electrode was placed on the surface of the muscle. Positive blue staining was observed in approximately 250 fibers which were localised to the middle third of the muscle. In (B) the fibers showed widepread staining, indicating transfection along a longer segment og the fiber and/or increased tra sgene expression.

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